International Journal of Organ Transplantation Medicine Comparison of Serum Neutrophil Gelatinase-associated Lipocalin (ngal) with Serum Creatinine in Prediction of Kidney Recovery after Renal Transplantation

AbsTrAcT Background: Because of some insult to kidney during transplantation, assessment of kidney function after the procedure is essential. It would be ideal to find a marker better than creatinine to early predict the acute kidney injury.


INTrOducTION
A cute kidney injury (AKI) is a common problem in many clinical settings including renal transplantation. Because of many limitations, indicators such as serum creatinine or urinary output are not reliable for early diagnosis of deteriorating renal function. A biomarker to facilitate prediction of kidney function would be valuable. Based on published articles, it seems that urinary and plasma neutrophil gelatinase-associated lipocalin (NGAL), are reliable biomarkers for the subsequent development of clinical AKI. However, there is not a consensus on this biomarker for prediction of graft dysfunction after transplantation [1][2][3][4].
Belonging to the lipocalin superfamily, NGAL, a 25-kDa protein was initially found in activated neutrophils. However, other cells, such as renal tubular cells, may release NGAL in response to inflammation or injury [5,6].
In a meta-analysis of 19 studies with 2538 patients, it has been documented that NGAL level increases in urine and/or plasma before clinical diagnosis of AKI with prognostic significance; it has also been shown to be a useful indicator to predict renal replacement therapy initiation and in-hospital mortality [7].
In kidney transplantation, Mishra, et al., examined 25 specimens of kidneys biopsies obtained at approximately one hour of reperfusion after transplantation for NGAL expression by immunohistochemistry and found a strong correlation between NGAL expression not only with cold ischemia time (r=0.87, p<0.001) but also with peak post-operative creatinine level days later (r=0.86, p<0.001) [3]. NGAL level in post-transplantation period has positive correlation with serum creatinine; the first day urinary NGAL predicts dialysis need within the first week of transplantation better than serum creatinine [2,8,9] Hollman, et al., in their study on 176 kidney recipients, also showed slower decrease in urinary NGAL compared to those without delayed graft function (DGF); urinary NGAL of the first posttransplantation day predicted DGF even when the clinicians did not expect which reflects the prognostic value of NGAL in these patients [10]. The value of NGAL was even highlighted in prediction of early functional recovery of transplanted kidneys from cardiac death donors in comparison with living donors [11].
The objective of this study was to re-evaluate the predictive value of serum NGAL in predicting DGF in kidney transplantation and figure out the best time for measurement of NGAL.

PATIeNTs ANd MeThOds
This prospective observational study was performed on 33 renal allograft recipients who received their kidneys in Imam Hospital, Tehran, Iran, from March 2010 to September 2010. The immunosuppressive regimen consisted of cyclosporine, mycophenolate mofetil, and prednisolone. The protocol of this study was approved by the Medical University Ethics Committee. Serum NGAL (Antibodyshop, Gentofte, Denmark) and creatinine, hemoglobin, blood glucose (commercially available kits) and urine output were measured at 0, 12, 24, 36, 48 and 72 hours after transplantation. Serum creatinine was measured by Jaffé method. Routinely, all patients underwent renal scintigraphy on the second day after operation. Measurements were performed by hospital laboratory personnel who were not aware of the NGAL source.
We used the definition of Hall, et al, for DGF (i.e., need to dialysis in the first week of transplantation), slow graft function (SGF, i.e., reduction in serum creatinine on day seven to the time of transplantation <70%), and immediate graft function (IGF, i.e., >70% reduction of serum creatinine on seventh day in comparison with transplantation time) [12].

Statistical analysis
Data were expressed as mean±SD (range). One-way analysis of variance (ANOVA) was used to assess the level of plasma NGAL at different hours in three levels of graft function. Kruskall-Wallis test was used to determine if the difference in cause of renal failure, sex of donor or recipient had any impact on renal function after transplantation. Repeatedmeasures ANOVA was used for plasma NGAL and creatinine levels at different time points. Correlations between NGAL and other variables were evaluated by Pearson's or Spearman's correlation coefficients, as appropriate. Receiver-operating characteristic (ROC) curve was used to determine the cutoff point for plasma NGAL level to predict DGF. All tests were two-sided; p<0.05 was considered statistically significant.

resulTs
The study was conducted on 33 kidney recipients. There were 16 men and 17 women. The mean±SD age of participants was 36.3±12.2 (range: 14-58) years. In 12 (36%) patients the cause of end-stage renal disease (ESRD) was unknown. Hypertension and diabetes were the cause in 11 (33%) and 4 (12%) patients, re-spectively. Four (12%) patients had preemptive transplantation and the rest had history of dialysis. The source of kidney was from braindead donors (BDD) in 20 (61%) patients.
Twenty-seven (82%) patients were prescribed polyclonal antibodies during induction therapy. The demographic data are shown in different groups in Table 1. Urine output after operation had a significant negative correlation with creatinine level at 12, 24, 48 and 72 hours of transplantation; however, its negative correlation lasts till 24 hours after transplantation with serum NGAL level. At any time, serum NGAL, and creatinine levels were significantly higher among patients with DGF and SGF compared with those with IGF (p=0.024 and 0.026, respectively). However, when they were compared between those who got IGF vs. non-IGF, serum creatinine levels were not significantly different (p=0.59) and serum NGAL levels differed significantly (p=0.02).
In order to assess the predictive value of NGAL vs. creatinine for DGF or non-IGF (combined DGF or SGF) we compared the areas under the ROC curves (AUCs) ( Table 3). Serum NGAL or serum creatinine on the first post-transplantation day had similar AUC and predictive value to predict the patient's need to dialysis in the first week. However, using

dIscussION
Diagnosis of acute graft dysfunction is usually made with increased serum creatinine concentration, which is a late signal of kidney injury. Nevertheless, the sooner treatment is initiated the better outcome is expected. The treatment  should be as close as possible to the insult just before the diagnosis is made by serum creatinine [13].
Our study, like other studies, confirmed that plasma NGAL level was more useful than absolute or percentage of serum creatinine decrease in predicting DGF [7,9,11,12]. However, plasma NGAL level before with first 24 hours of transplantation was not able to differentiate patients with DGF from those with non-DGF.
In this study, we found that the best time for measuring plasma NGAL level for such purpose is 24 hours after the transplantation. Similar to Lebkowska, et al., we found a significant decrease in serum NGAL as early as one day after transplantation-before the serum creatinine level decreases [2]. Bataille, et al., in 41 transplant patients found an early and significant post-operative decrease in plasma NGAL after 12 hours of transplantation in the IGF group, which remained elevated in the DGF group throughout the follow-up period [14]. Similar to our study, they showed that the decrease in creatinine level was delayed compared with plasma NGAL decrease.
Mishra, et al., found the highest NGAL staining intensity in biopsies of one hour of reperfusion after transplantation in those with DFG [3]. Although plasma NGAL level after transplantation was highest in DGF group, surprisingly, no significant correlation was observed between NGAL and DGF, which may be due to limited sample size or its excretion into the urine rather than being released into the circulation. It was shown that the urine output after operation negatively correlated with plasma NGAL after operation which supports the explanation.
The main limitation of our study was the small  sample size (20 from BDD) for which we could not do subgroup analysis. However, based on prior studies, the results support the finding that plasma NGAL level, especially after 24 hours of transplantation, is an early accurate predictor of both the need to dialysis and slow graft function within the first week of kidney transplantation.